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US 20200383331A1 IN (19United States ( 12 ) Patent Application Publication ( Pub. No.:USQO2Q/QZ8333l Al HEINRICHER ( 43 ) Pub . Date : Dec. 10 , 2020 ( 54 ) COMPOSITIONS AND METHODS FOR AOIN 43/40 ( WQOQI LARGE - SCALE IN VITRO PLANT AOIN 43/08 ( 2006.01 ) BIOCULTURE A01N 37/52 ( 2006.01 ) AOIN 4730 ( 2006.01 ) ( II ) Applicant: BQOSHIQOQT LLC , Hailey, IDUS A016 22/15 ( WQGOI ( 52 ) U.S. CI . ( 72 ) Inventor: Jackie HEINRICHER , Anacortes , WA CPC AOIN 43/90 ( 2013.01 ) ; AO1G 31/00 (US ) ( 2013.01 ) ; A01N 59/08 ( 2013.01 ) ; A01N 59/20 ( 2013.01 ) ; A01N 59/16 ( 2013.01 ) ; ( 21 ) Appl . No .: 16 /728,478 A01N 59/14 ( 2013.01 ) ; A01N 31/06 ( 2013.01 ) ; A01N 43/78 ( 2013.01 ) ; A01N ( 22 ) Filed : Dec. 27 , 2019 37/10 ( 2013.01 ) ; A01N 43/82 ( 2013.01 ) ; AOIN 59/12 ( 2013.01 ) ; AOIN 37/44 Related U.S. Application Data ( 2013.01 ) ; A01N 43/40 ( 2013.01 ) ; A01N ( 63 ) Continuation of application No. PCT /US2018 / 43/08 ( 2013.01 ) ; A01N 37/52 ( 2013.01 ) ; 040637 , filed on Jul. 2 , 2018 , Continuation of appli AOIN 47/30 ( 2013.01 ) ; A01G 22/15 cation No. PCT/ US2018 / 040646 , filed on Jul. 2 , ( 2018.02 ) ; A01N 59/00 ( 2013.01 ) 2018 . ( 60 ) Provisional application No. 62 / 527,946 , filed on Jun . ( 57 ) ABSTRACT 3Q , provisional application No. 62 /6II , & a , The present invention provides media , kits , systems , and filed on Dec. 29 , 2017 , provisional application No. methods for achieving large scale pistachio production 62 / 527,862 , filed on Jun . 30 , 2017 . within a short time via bioculture , large scale yam produc tion within a short time via bioculture, high multiplication Publication Classification rate of plants including cannabis via in vitro micropropaga ( 51 ) Int . Ci . tion , high induction rates of somatic embryos from later A01N 43/90 ( 2006.01 ) buds in bamboo , reduced production of phenolic compounds AOIN 59/00 ( 2006.01 ) in plants, high production of virus - free plants , including AOIN 59/08 ( 2006.01 ) potato , and large scale hemp production via culturing. The AOIN 59/20 ( 2006.01 ) present invention for pistachio and yam production results in AOIN 59/16 ( 2006.01 ) shorter tuber development phase and higher yield . In some AOIN 59/14 ( 2006.01 ) embodiments , the present invention provides compositions, AOIN 31/06 ( 2006.01 ) methods, and systems for the micropropagation and mass AOIN 43/78 ( 2006.01 ) production of perennials, grasses, bamboos, cannabis and AOIN 37/10 ( XQOOQI phyto - pharmaceutical plants as well as hemp plants . In some AOIN 43/82 ( 2006.01 ) embodiments , the present invention provides compositions, AOIN 59/12 ( 2006.01 ) methods , and systems for reducing the production of a AOIN 37/44 ( 2006.01 ) phenolic by a plant , such as bamboo . 200 210 12 Patent Application Publication Dec. 10 , 2020 Sheet 1 of 39 US 2020/0383331 A1 1cm Shoottipnecrosisinpistachioplantculturedvitro(B0012Medium) 2FIG.1 Patent Application Publication Dec. 10 , 2020 Sheet 2 of 39 US 2020/0383331 A1 1cm Newshootsdevelopedfromaxillarybudsofsingle-nodeexplantspistachioafter3days ofcultureon(B003Medium) FIG.2 Patent Application Publication Dec. 10 , 2020 Sheet 3 of 39 US 2020/0383331 A1 Rootdevelopmentinpistachio(PistaciaatlanticaxP.intergerrina)plantsculturedonrootingmediumB0018 for2weeks.Theimageontherightisaclose-upofleftphotoarrowspointtonewroot FIG.3 Patent Application Publication Dec. 10 , 2020 Sheet 4 of 39 US 2020/0383331 A1 3 ? plantsgrowinginvitroonB003mediumWell-developed,healthypistachio { FIG.4 Patent Application Publication Dec. 10 , 2020 Sheet 5 of 39 US 2020/0383331 A1 -190 -150 Controller SecondMedia Vessel 110 Growth Vessel GasSupply 170 MediaFirst Vessel 100 130 FIG.5 Patent Application Publication Dec. 10 , 2020 Sheet 6 of 39 US 2020/0383331 A1 20 52 210 ***** go 0 **2-2 12 216 CO IKI 200 FIG.6 Patent Application Publication Dec. 10 , 2020 Sheet 7 of 39 US 2020/0383331 A1 S02 297. 232--- 967 05rose FIG.7 Patent Application Publication Dec. 10 , 2020 Sheet 8 of 39 US 2020/0383331 A1 Ht2 www.240 952 PL 2412 FIG.8 Patent Application Publication Dec.10.2020 Sheet 9 of 39 US 2020/0383331 A1 -222 22 FIG.9C REE 922 ??? FIG.9A @ FIG.9B CC 2 Patent Application Publication Dec. 10 , 2020 Sheet 10 of 39 US 2020/0383331 A1 320 326 322 324 complete? Yes complete? Yes Executethirdoperatingmode ?second incubation sequence isplant propagation sequence Endplantpropagationsequence No No NO Executethirdoperatingmode: complete? Yes Startplantpropagationsequence Startfirstincubationsequence Establishfluidcommunication betweenfirstmediacontainerand vesselgrowth Executefirstoperatingmode firstis incubation sequence Startsecondincubationsequence Establishfluidcommunication betweensecondmediacontainerand vesselBrowth Executesecondoperatingmode 312 302 304 306. 308 310 314 316 318 FIG.10 Patent Application Publication Dec. 10 , 2020 Sheet 11 of 39 US 2020/0383331 A1 MSorB5 BOO22 tuberization MS 1 80,000 900 MSorB5 BO021 tuberization MS 0.02 60,000 900 pre-tuberization MSorB5 tuberosumL.)tuberizationofpotato(SolanumvitropropagationandCompositionmediaforin BO019 MS 0.5 30,000 900 MurashigeandSkoogmediumsalts(1962)concentrations*Allaregivenining/l;thermolabilecoinpoundswere Gamborgetal.(1968)Vitaminmixtureasdescribedby cooledto40-50°Ctheautoclavedmediafilter-sterilizedandadded tuberizationpre- MSorB5 BO018 MS 1 4 30,000 900 plantpropagation MSorB50 BO017 MS" 20,000 900 5,000 Growthregulators: Carbohydrate: Bufferingagent: Solidifyingagent: Macro-andmicronutrients Ancymidol Agar Components Vitamins IAA NAA 2iP Sucrose MES 5.8pH FIG.11 Patent Application Publication Dec. 10 , 2020 Sheet 12 of 39 US 2020/0383331 A1 MSorB5 BO027 tuberization MS 5 80.000 900 MSorB5 BOO26 tuberization MS 0.5 80,000 900 MSorB5 Compositionofadditionalmediaforinvitrotuberizationpotato(SolanumtuberosumL.) BOO25 tuberization MS 0.02 0.5 80,000 900 MurashigeandSkoogmediumsalts(1962)concentrations"Allaregiveninmg/l;thermolabilecompoundswere Gamborgetal.(1968)mixtureasdescribedby 40-50CVitaminmediacooledtoaddedtheautoclavedfilter-sterilizedand tuberizationpre- MSorB5 BOO24 MS 5 30,000 900 tuberizationpre- MSorB50 BOO23 MS 1 4 0.5 30,000 900 Macro-andmicronutrients Growthregulators: Carbohydrate: Bufferingagent: Solidifyingagent: IAA 2iP Ancymidol MES Agar Component Vitamins NAA Sucrose 5.8PH FIG.12 Patent Application Publication Dec. 10 , 2020 Sheet 13 of 39 US 2020/0383331 A1 FIG . 13 Copposed BO031 BO048 BO049 DKW BOO51 B0050 BO052 BO053 0 0 0 2 0 0 Patent Application Publication Dec. 10 , 2020 Sheet 14 of 39 US 2020/0383331 A1 MMMM FIG.14 Patent Application Publication Dec. 10 , 2020 Sheet 15 of 39 US 2020/0383331 Al AA/ A//// : FIG.15 Patent Application Publication Dec. 10 , 2020 Sheet 16 of 39 US 2020/0383331 A1 FIG.16 Patent Application Publication Dec. 10 , 2020 Sheet 17 of 39 US 2020/0383331 A1 FIG.17 Patent Application Publication Dec. 10 , 2020 Sheet 18 of 39 US 2020/0383331 A1 NIINNNINAINIAI ***** WW *** FIG.18 Patent Application Publication Dec. 10 , 2020 Sheet 19 of 39 US 2020/0383331 A1 FIG.19 FIG.20 Patent Application Publication Dec. 10 , 2020 Sheet 20 of 39 US 2020/0383331 A1 0 FIG.21 Patent Application Publication Dec. 10 , 2020 Sheet 21 of 39 US 2020/0383331 A1 FIG.22 Patent Application Publication Dec. 10 , 2020 Sheet 22 of 39 US 2020/0383331 A1 FIG.23 Patent Application Publication Dec. 10 , 2020 Sheet 23 of 39 US 2020/0383331 A1 | FIG.24 Patent Application Publication Dec. 10 , 2020 Sheet 24 of 39 US 2020/0383331 A1 FIG.25 Patent Application Publication Dec. 10 , 2020 Sheet 25 of 39 US 2020/0383331 A1 FIG . 26A Chemical mg/ l 80054 B0055 BOO56 BO057 BO058 BOOS9 B0060 B0061 INH.NO 1,650.00 1,650.00 1,650.00 1,650.00 1,650.00 1.650.00 1,650.00 NH .( SO ) 134,00 Ca (NO3 ) 24H20 950.00 2,500.00 950.00 950.00 950.00 950.00 950.00 950.00 MgSO4.7H , 0 370.00 250.00 370.00 370.00 370.00 370.00 370.00 370.00 MnSo..4,0 16.90 10.00 16.90 16.90 16.90 16.90 16.90 16.90 ZnSO4.7H20 8.60 2.00 8.60 8.60 8.60 8.60 8.60 8.60 C450.5H0 0.03 0.03 0,03 0.03 0.03 0.03 CaCl2240 440.00 150.00 440.00 440.00 440.00 440.00 440.00 440.00 KI 0.83 0.75 0.83 0.83 0.83 0.83 0.83 0.83 CoCl2.6H 0 0.03 0.03 0.03 0.03 0.03 HBO 6.20 6.20 6.20 6.20 6.20 6.20 6.20 Na Mo09.2H2O 0.25 0.25 0.25 0.25 0.25 0,25 0.25 0.25 K2SO4 KH PO 170.00 170.00 170.00 170.00 170.00 170.00 170.00 FeSO..7H20 27.80 27.80 27.80 27.80 27.80 27.80 27.80 27.80 Na EDTA 37.30 37.30 37.30 37.30 37.30 37.30 37.30 37.30 Myo -Inositol 100.00 100.00 Thiamine HCI 10.00 10.00 10.00 10.00 10.00 10.00 10.00 Pyridoxine HCI 1.00 1.00 1.00 Nicotinicacid 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Glycine 2.00 2.00 2.00 2.00 2.00 Ribavirine (Virazole ) 2.00 1.50 2.50 2.50 BAP 0.001 to 10,0 0,001 to 10,0 0,001 10 10.0 0.001 to 10.0 10.001 to 10.0 0.001 to 10.0 0.001 to 10.0 0.001 to 10,0 TOZ Meta - Topoline 0.01 to 5.0 0.01 to 5.0 0.01 to 5.0 0.01105.0 0.01 to 5.0 0.01 to 5.0 0.01 to 5.0 0.01t05.0 1.00 JAA 21p 3,00 3.00 Paclobutrazo GAZ Citric acid 10.00 Glutamine 100.00 Casein hydrolysate 100.00 Sucrose 30,000.00 30,000.00 3,000 to 10,000 3,000 to 10,000 30,000.00 30,000.00 Charcoal 5,000.00 1,000.00 Agar 5,000,00 5,000.00 5,000.00 5.000.00 5,000.00 5,000.00 3,000.00 3,000.00 Carregeenan 5.50 $5.50 5.50 5.50 5.50 5.50 5.80 5.80 Patent Application Publication Dec.